PROJECT SUMMARY Progressive hearing loss is a prevalent health problem that often stems from the loss or dysfunction of sensory hair cells in the inner ear. Hair cell function depends on actin-based protrusions called stereocilia to detect the physical movement of sound. Functional stereocilia must be maintained for the life of an organism since hair cells are not renewed or regenerated. Thus, stereocilia homeostasis is critical for continued auditory function. Tip links transmit force from stereocilia deflection to gate associated mechanotransduction channels found at stereocilia tips. Rows of stereocilia that normally have active channels also have more actin incorporation at their tips. In addition, mutations in the Cdh23 gene, which encodes the tip link component cadherin-23, strongly influence progressive hearing loss and stereocilia length maintenance in mouse models. Together, these data suggest that tip links or mechanotransduction regulates actin dynamics in stereocilia. Recently, we and others found that stereocilia actin cores are highly stable, except at stereocilia tips, where there is more dynamic actin turnover. However, several unanswered questions remain including whether actin binding proteins in stereocilia are similarly stable, how the stereocilia actin core is made to be stable and whether actin core instability directly influences progressive hearing loss. It is also unclear how the dynamic region of actin at stereocilia tips is regulated and if it contributes to stereocilia length maintenance. To address these questions, we will use transgenic reporters to monitor the dynamics of actin and the actin crosslinker fascin-2 in vivo and ex vivo, both in normal mice and in mice with deafness causing mutations in actin binding proteins found in stereocilia. We also hypothesize that stereocilia stability is particularly critical for stereocilia maintenance when tip links break. This model will be tested by measuring actin incorporation as Cdh23 expression is varied. If stereocilia shorten following tip link loss, then actin polymerization would be required to extend stereocilia back to their original length. We have found that the actin severing proteins destrin and cofilin localize to stereocilia tips, and we will determine if they promote new actin incorporation within the dynamic tip zone. Together, these experiments will provide critical insights into the roles of both extraordinarily stable and more dynamic regions of the actin core in maintaining functional stereocilia.